1. Field of the Invention
The present invention relates to a method for detecting, distinguishing or counting cells or microorganisms in a live state at a nucleic acid level by the use of a probe comprising a nonnatural type nucleic acid.
2. Related Background Art
In recent years, it has become important to specify, distinguish, detect or count cells, microorganisms or the like (hereinafter referred to as "target individuals") at a gene level, i.e., at a nucleic acid level for the sake of the diagnosis of a hereditary disease based on the variation of a gene, the detection of cancer cells by the expression of a carcinogenic gene or the detection of a genetic recombinant at a research level.
Heretofore, for the detection of the target individuals and for the count of the number of the target individuals, there have been used a light field microscope observation method, a phase difference microscope observation method, a differential interference microscope observation method and so forth. In order to solve the problem that the target individuals are difficult to observe, or in order to more effectively or more highly sensitively detect the target individuals, they have usually been stained with a dyestuff such as Methylene Blue or Safranine, or a fluorescent dyestuff such as Auramine or fluorescein, and then observed by a light field microscope or a fluorescent microscope.
Needless to say in the case that the target individuals are not stained and even in the case where they are all or partially stained, they can be distinguished from other kinds of individuals by observing and evaluating them from the standpoint of morphology, but a difference of genetic variation cannot be distinguished between the same kind of individuals having no difference in morphology. In addition, the dyestuff and the fluorescent dyestuff scarcely permeate the cytoplasmic membrane and the nuclear membrane of the target individuals in a live state (live target individuals), and so it is difficult to detect the live target individuals with the aid of staining.
On the other hand, as a technique for evaluating the target individuals at the gene level or the nucleic acid level, there has been recently carried out a method in which nucleic acid is extracted from the target individuals and then its base sequence is inspected or patterns obtained by cutting the nucleic acid with a restriction enzyme are inspected. However, even according to such a method, it is still impossible to evaluate the target individuals in the live state, while the morphology is maintained. Furthermore, in the test in which the nucleic acid from the target individuals is treated, the series of operations are intricate and a long period of time is inconveniently taken for the detection.
As one technique for solving the problems of the conventional methods, an in situ hybridization method by which the target individuals can be evaluated at the gene level has been researched in recent years, and it has been partially put to practical use [e.g., Nucleic Acid Research, Vol. 20, No. 1, pp. 83-88 (1991)]. Next, the in-situ hybridization method will be briefly described.
(1) A nucleic acid probe having a base sequence which is complementary to a base sequence inherent in a nucleic acid in the target individuals is prepared. This nucleic acid probe has a certain detectable site (label).
(2) The target individuals are killed with methanol or the like, and then fixed.
(3) The nucleic acid probe is hybridized with the nucleic acid of the fixed target individuals in the target individuals (in-situ).
(4) The probe which has not formed a hybrid is washed off.
(5) The hybrid formed in the target individuals is detected by the utilization of the above-mentioned label.
According to this in-situ hybridization method, a step of extracting the nucleic acid from the target individuals can be omitted. In addition, the detection of the target individuals at the gene level or the nucleic acid level is possible, while the morphology is maintained, and the number of the target individuals can also be counted. In this method, the live target individuals are killed, whereby the cytoplasmic membrane and the nuclear membrane are modified to facilitate the passage of the nucleic acid probe through these membranes.
As described above, according to the in-situ hybridization, the target individuals can be distinguished, detected or counted at the nucleic acid level, while maintaining their morphology, which has been impossible by conventional methods. However, there still remains the problem that the target individuals cannot be detected in the live state.
The reasons why the live target individuals cannot be detected by the conventional in-situ hybridization can be summarized as follows:
(1) The nucleic acid portion of the nucleic acid probe is of a natural type regardless of what is obtained from nature or chemically synthesized, and so the hydrophilic nature of a phosphorus portion is strong. Thus, the probe scarcely permeates the cytoplasmic membrane and the nuclear membrane of the live target individuals.
(2) Even if the nucleic acid probe permeates the cytoplasmic membrane and the nuclear membrane, it is degraded by a single-strand nucleic acid degradation enzyme in the live target individuals on occasion. This tendency is larger when the nucleic acid portion of the nucleic acid probe is RNA than when it is DNA.
(3) In the case that the nucleic acid portion of the nucleic acid probe is DNA, even if the nucleic acid probe forms a hybrid together with the target nucleic acid in the live target individuals, the hybrid is degraded by a double-strand nucleic acid degradation enzyme in the live target individuals on occasion.
(4) Some of fluorescent dyestuffs which can usually be used as the label portion of the nucleic acid probe are harmful to the live target individuals, and if such a dyestuff is utilized, the live target individuals die on occasion.